Medicinal Chemistry

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Future

Research Article

Medicinal Chemistry

to & ers,

Searching phase II enzymes inducers, from Michael acceptor-[1,2]dithiolethione hybrids, as cancer chemopreventive agents

Marcos Couto1, Stefani de Ovalle1, Mauricio Cabrera1, Hugo Cerecetto*,1 & Mercedes González1 Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, 11400 Montevideo, Uruguay *Author for correspondence: [email protected]

1

or

Pr

oo

f

Background: Cancer chemoprevention involves the carcinogenic process prevention, delay or reverse by the administration of chemopreventive agents, which are able to suppress or block the carcinogen metabolic activation/formation. The increased activity of phase II detoxification enzymes such as quinone-reductase (QR) and glutation-S-transferase (GST) correlates with the protection against chemicallyinduced carcinogenesis. It has been shown that synthetic chalcones and 3H-[1,2]dithiole-3-thiones promote expression of genes involved in chemoprevention. Materials & Methods: Herein, the induction of phase II enzymes by designed Michael acceptor-dithiolethione hybrids was studied. Results & Discussion: Hybrids 5 and 7 displayed the induction of quinone-reductase and glutation-S-transferase in vitro in the same order on the wild-type mouse-hepatoma Hepa 1c1c7 and on the arylhydrocarbon-nuclear-translocator (Arnt)-defective mutant BPrc1 cells indicating that 7 displays the best chemopreventive potential.

bility to react with DNA and proteins promoting damages and cellular disruption that could initiate carcinogenic process. However, phase II enzymes promote the xenobiotics conjugation with endogenous ligands, like glutathione and glucuronic acid, to facilitate their excretion to diminish their potential carcinogenic effects. Examples of phase II enzymes are UDP-glucuronosyltransferase, NAD(P)H: quinone reductase (QR, EC 1.6.5.2), and glutathione S-transferase (GST, EC 2.5.1.18) [5] . QR is considered a phase II enzyme because it has protective functions, is induced coordinately with other phase II enzymes and is regulated by enhancer elements similar to those that control GST [6] . Studies have shown that increase in phase II enzyme activity, such as QR and GST, correlates with the protection against chemicalinduced carcinogenesis in the initiation and promotion stages [1,2,6] . The enzyme inducer CCAs are of two types: monofunctional and bifunctional. Monofunctional inducers (pathway green in Figure 1) increase selectively phase II enzymes activating the antioxidant response element

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Carcinogenesis is a complex and protracted multistage process initiated by events wherein an endogenous or exogenous agent damages a cellular macromolecule. Cancer chemoprevention involves prevention, delay or reverse of the carcinogenic process through administration of drugs named as cancer chemopreventive agents (CCAs). These CCAs are able to suppress the carcinogen metabolic activation or to block the formation of ultimate carcinogens [1] . CCA could interfere with the carcinogenic process at various levels by blocking initiation and by suppressing later stages involving promotion, progression, angiogenesis, invasion and metastasis [2] . The possible ways to block the initiation of carcinogenesis could include metabolism alteration of procarcinogens in favor of conjugation and excretion of reactive metabolites [3] , regulated by phase I and phase II enzymes [4] . Phase I enzymes, for example, Cyt P450 (CYP), Cyt b5 and NADPH-cytochrome c reductase, involves xenobiotics oxidation, reduction and hydrolysis processes and the end products of these processes are mainly electrophilic entities with enhanced capa-

10.4155/FMC.15.32 © 2015 Future Science Ltd

Future Med. Chem. (2015) 7(7), 857–871

part of

ISSN 1756-8919

857

Research Article  Couto, de Ovalle, Cabrera, Cerecetto & González

Bifunctional inducer

Ahr Arnt

CYP1A CYP1B

XRE ARE

Phase II

f

XRE

Keap1

or

Pr

Nrf2

oo

Phase II

Monofunctional inducer

Figure 1. Model of the relationship between the mechanism of action of monofunctional and bifunctional inducers of phase II enzymes and cancer chemopreventive agents .

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(ARE) through the Keap1-Nrf2 system. While the bifunctional inducers (pathway red in Figure 1) increase both phase I and phase II enzymes by binding to the Ah receptor (Ahr) and then the Ahr-ligand complex is translocated to the nucleus, through the Ah nuclear translocator receptor (Arnt, Figure 1), activating the xenobiotic response element (XRE). Many structurally unrelated agents including the polyaromatic heterocycles, barbiturates, phenolic antioxidants, curcuminoids, cinnamates (i.e.,[I] Figure 2A)  [7] , isothiocyanates, [1,2]dithiole-3-thiones (i.e., oltipraz, [II] Figure 2A), lactones, thiocarbamates and flavonoids were found to induce genes of phase II enzymes (Figure 2A)  [8–10] . Specifically, flavonoids are among the most studied CCA, being the natural ones the best-characterized, in other words, the flavanol quercetin ([III], Figure 2A) [11] . Chalcones, bioprecursors of flavonoids bearing the Michael-acceptor structure into a 1,3-diphenyl-2-propen-1-one system, have been studied by our group, finding excellent oral in vivo activity increasing liver phase II (QR and GST) enzyme activity and decreasing phase I (CYP1A1/CYP1A2, EC 1.14.14.1) ones [12] . Specifically, chalcones (IV–VII)

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Future Med. Chem. (2015) 7(7)

displayed the best behaviors in our in vivo tumorigenesis/chemopreventive and acute-toxicity studies resulting monofunctional inducers (Figure 2B) . Additionally, an in vitro model, using human mammary adenocarcinoma cells (MCF-7) [13,14] , was used to predict the in vivo activity. The fascinating biological properties of 3H-[1,2]dithiole-3-thione system have attracted the attention of numerous researchers [15,16] . Among the different bioactivities, 3H-[1,2]-dithiole-3-thiones have been described as cancer chemopreventive drugs (i.e., oltipraz, (I) Figure 2A) [17,18] . In some instances, the mechanism of action has been associated to the release of H2S by the dithiolethione heterocycles [19] . While in other cases more complex processes have been proposed, in other words, the activation of Nrf2 signaling and induction of phase II enzymes undergo reductive cleavage, resulting in the generation of ROS [20] , or not [21] . In this context and as a continuation of our previous work, we are currently interested in the development of hybrid CCAs which combine Michael-acceptor framework and 3H-[1,2]dithiole-3-thione moiety (Figure 2C) to improve the desired biological activity.

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Research Article

Searching Phase II enzymes inducers, from Michael acceptor-[1,2]dithiolethione hybrids, as CCA 

OH OH H O

S S

H O

Oltipraz, (II)

OH

O (IV)

O

O

O

oo

(VI)

QR , GST=, CYP

-connector-

QR , GST=, CYP

Pr

A-

Quercetin (III)

O

(V)

QR , GST , CYP

A

B

Br

O (VII)

QR , GST , CYP

B-

S

or

Ar-

OH

ArS S or -connector-

O

or

O

f

OH

S

OH OH

(I)

S S

O

N

S

H

HO

N

Ar- = Ph- or o-(OH)Ph-

A ut h

Figure 2. Cancer chemopreventive agents. (A) Examples of chemopreventive agents. (B) Chalcones described by our group as monofunctional phase II enzymes inducers. (C) Hybrid Michael acceptor dithiolethiones.

Herein we describe the development of hybrid Michael acceptor-dithiolethiones (Figure 2C) which together with synthetic intermediates or starting materials were evaluated as monofunctional enzymatic inducers. We determined QR and GST phase II enzymes activities in an in vitro model. We use wildtype mouse hepatoma Hepa 1c1c7 and aryl hydrocarbon nuclear translocator (Arnt)-defective mutant BPrc1 cells [22,23] . Additionally, to determine the role of the double bond in the bioresponse we developed a non-α,β-unsaturated carbonyl-derivative. For the most relevant derivatives the concentrations for doubling enzymatic activity (CDs), and chemopreventive indexes, CIs (ratio between IC50 and CDs), were determined. Besides, stabilities of the selected derivatives in aqueous solution at different pHs, in biological milieu and in plasma were studied in order to confirm their potentials as novel cancer chemopreventive drugs. The structure-activity analysis showed that the hybrid structure, Michael acceptor and [1,2]dithiole systems, as well as the absence of some moieties would be took into account for further structural modifications.

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Results & discussion For the synthesis of hybrid derivatives 5 and 6 (Figure 3) , we prepared the chalcone intermediates 1 and 2 using previously described methodology [24] . Then they were coupled, using DCC/DMAP [25] , with 5-(4-hydroxyphenyl)-3H-[1,2]dithiole-3-thione, 4, generated from anethole in two steps [26] . The same procedure was used to prepare derivative 7 (Figure 3) , which contains the Michael-acceptor cinnamyl framework, and derivative 8. The last, the nonα,β-unsaturated carbonyl-derivative, was developed to identify the relevance of the unsaturation in the studied biological activity. Attempts to reduce the double bond of derivatives 5–7, by catalytic hydrogenation, were unsuccessful. All the efforts produced modification of the other moieties. Apart from derivative 7, where the [1,2]dithiole-3-thione-5-yl-moiety occupies the β-position (Figure 2C), we designed derivatives 10 and 11 (Figure 4A) . The intermediate 9 was obtained, in very low yield, via nucleophilic substitution using the phenol 4 as starting material (Figure 4A) . When the aldolic condensations between 9 and benzaldehyde

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Research Article  Couto, de Ovalle, Cabrera, Cerecetto & González

CO2H

KOH/MeOH

+ O

O

CHO

DCC/DMAP

S

(HO)n

O

S

145°C

HCl

OR

-R = -CH3, 3 4

-R = -H,

f

215°C S S

OH

O

n = 0, 5 n = 1, 6

S

S

N

OH

S

S

O

DCC/DMAP

O

O

S S

OH S

O

DCC/DMAP

7

S

S

OH

S

Pr

O

S

N S

O

O

O

n = 0, 1 n = 0, 2

O

S8 /

(HO)n

HO

oo

(HO)n

O

8

S

S

or

Figure 3. Synthetic procedures used to prepare the studied hybrid derivatives 5–7 and alkyl derivative 8. DCC: Dicyclohexylcarbodiimide DMAP: 4-(N,N-dimethylamino)pyridine.

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or salicylaldehyde were performed, not desired products were generated in mild conditions (see examples in Table 1). However decomposition of 9, recovering phenol 4, was observed in strong conditions (see example in Table 1). This result, together with the low yields in the synthesis of derivatives 5, 6 and 9 showed both the poor nucleophilic and good leaving group characters of the phenolate of 4. This could probably be the result of the excellent delocalization of the negative charge to the [1,2]dithiolethione system. Attempting to improve the yield of intermediate 9, we probed some other reaction conditions. In one specific condition (Figure 4B) we were able to produce the (E)3-[1-(alkylthio)propylidene]-3H-[1,2]dithiole 12 as the main product [27] . This kind of serendipitous product was previously prepared in other conditions [28,29] but was not studied as CCA. To augment the chemoand bio-diversity we also assayed this procedure using other reactants, in other words, the dithioletione 3 and phenacyl bromide, generating another [1,2]dithioles, in other words, 13, 15 and 17 (Figure 4B) . Additionally, in these reactions the product 14 and the iodide salts 16 and 18 were also isolated [30] and they were included in the biological studies. All the new compounds, 5–8, were characterized by 1H-NMR, 13CNMR, COSY, HMQC, and HMBC experiments, and

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Future Med. Chem. (2015) 7(7)

MS. The compounds, according to H–H coupling constants and NOE-diff experiments, were obtained as the E-isomer around the alkenic moiety. The purity of the synthesized compounds was established by thin layer chromatography (TLC) and microanalysis. Only compounds with analytical results for C, H and N within ±0.4 of the theoretical values were considered pure enough. All the compounds, 1–9, and 12–18, were tested in vitro for their capabilities to induce exclusively phase II enzymes. For that, a model that uses two different cellular systems were employed, in other words, wild-type Hepa 1c1c7 and mutant BPrc1 cells. The increment of the enzymatic activity in the mutant cell line concomitant with the increment in the wild type indicates that the compound tested is a monofunctional inducer (induces only phase II enzymes, Figure 1). When the increment is only in the wild-type, the compound is considered as a bifunctional inducer (induction of phase I and phase II enzymes, Figure 1)  [22] . According to this expected biological behavior we used as inducer-descriptor the ratio of specific activity between wild and mutant cells, in other words rH/B, where a value near to 1, and the concomitant increment of specific activity, indicates a monofunctional behavior while a value higher than 1, and the concomi-

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Research Article

Searching Phase II enzymes inducers, from Michael acceptor-[1,2]dithiolethione hybrids, as CCA 

(OH)n O

S HO

Cl

S

S O

CaCO3/18-c-6/THF S benzyl dimethyl octyl amonnium chloride

4

CHO

S

(OH)n

O

S O

S

S

O

S

n = 0, 10 n = 0, 11

9

O

(1 equiv)

Cl

S

S

O

KI (17 equiv) K2CO3 (17 equiv) acetone reflux / 3 h

O

O

S

12

HO

Br

S

(1 equiv)

S

Ph

HO

S KI (17 equiv) K2CO3 (17 equiv) acetone reflux / 3 h

4

oo

O

S

f

O

S

O

Ph

S

(1 equiv)

Cl

S

O

O

S S

S S 14

Ph

O

S

O

S S

I S

15

16

+

-

O

O

O

Br

(1 equiv) Ph

A ut h

3

13

+

or

S

KI (17 equiv) K2CO3 (17 equiv) acetone reflux / 3 h

S

S

O

O

Pr

O

O

+

Ph

KI (17 equiv) K2CO3 (17 equiv) acetone reflux / 3 h

O

S S

S

O +

Ph S O

17

O

O I

S

18

S + O

Ph O

Ph

Figure 4. Synthetic procedures proposed and used to prepare the hybrid derivatives 10–18. (A) Designed hybrids 10 and 11. (B) Synthetic procedures used to prepare no initially designed compounds 12–18. THF: Tetrahydrofurane.

tant increment of specific activity indicates a bifunctional behavior. As a primary screening QR activities were determined in both cellular models analyzing the compounds at 10.0 μM (Table 2) . In this study, 4′-bromoflavone (4BFV) was used as control finding that it displays the typical behavior of a bifunctional inducer (Table 2), in other words, high QR expression in wild-type cells with a rH/B of 3.0. The compounds studied displayed a wide variety of results. The starting materials, the de-hydroxychalcone 1 and the [1,2] dithiolethione 3, displayed a monofunctional profile (rH/B of 0.7 and 0.6, respectively). On the other hand, the [1,2]dithiolethione 4 was clearly bifunctional displaying higher QR specific activity on the wild-type

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cells (rH/B = 2.0). The hybrid derivatives, 5 and 7, were monofunctional inducers (rH/B of 0.9 and 1.1, respectively) sharing this behavior with the parent compound 1. Moreover, the cinnamyl derivative 7 appears as the best monofunctional inducer because it almost reached the duplicate of the values of QR specific activity, respect to untreated cells, in both cellular systems. Given that the cinnamic acid was inactive in this kind of assay, according to previous description [31] . And that the phenol 4 clearly was a bifunctional inducer the association of these two frameworks, in the hybrid agent 7, resulted very successful. Additionally, the relevance of the α,β-unsaturated carbonyl-moiety was proved with the poor activity of the derivative 8, little

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Research Article  Couto, de Ovalle, Cabrera, Cerecetto & González

Table 1. Results of the reaction between methylketone 9 and benzaldehyde. Run

Base

Condition

Time (h)

Results†

a

K 2CO3 (1eq)

r.t.

72

(-)

b

K 2CO3 (1eq)

Reflux

12

(-)

c

KOH (1eq)

r.t.

3

(-)

d

KOH (1eq)

Reflux

4

4‡

(-): No reaction. Besides unreacted aldehyde. r.t: Room temperature. † ‡

f

products 12 and 16, and one bifunctional inducer derivative, 18, the CIs were determined (Table 3) . The CI is defined as the ratio of the concentration which inhibited the growth of cell lines by 50% (IC50 ) and the concentration that double QR specific activity in the same cell line (CD). Also, in this study 4BFV and t-butylhydroquinone (t-BHQ) were included as controls. The CIs revealed that hybrid compound 7 was the best new developed potential cancer chemopreventive agent with clear monofunctional inducer capacity. It possessed, in both cellular models, lower values of CDs between the different studied compounds. The cinnamate 7 displayed lower CD on Hepa 1c1c7 than [1,2]dithiolethione intermediate 3 with a CD near to 30 μM [31] , or similar CD on Hepa 1c1c7 as previously described cinnamates [32] . In this previous study the best was methyl 2′-hydroxycinnamate with CD of 15 μM. In addition, the hybrid derivative 5 and the salt 16 were moderate monofunctional inducers and in a less amount could be considered as CCAs. An additional interesting aspect of these new Michael acceptor-[1,2]dithiolethione hybrids to be highlighted

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or

Pr

oo

increment on the wild cells and absence of induction in the mutant ones. The hybrid derivative 6 displayed similar bifunctional inducer behavior as the parent compound, hydroxychalcone 2. Furthermore, the intermediate 9; the secondary products 14, 16 and 18; and the unexpected products 12, 13, 15 and 17 (Table 2) displayed very diverse behavior. On one side, derivative 9 did not produce effect on the QR specific activity, absence of induction in both cellular systems. Second, some of them had a clear bifunctional characteristics, in other words, the [1,2]dithioles 13, and 15 (rH/B of 2.4 and 2.3, respectively). Third, the [1,2] dithiolethiones 14 and 17 and the salt 18 acted as weak bifunctional inducers (rH/B of 1.4, 1.7 and 1.8, respectively). Four, the [1,2]dithiole 12 and the salt 16 were monofunctional inducers (rH/B of 1.1 and 0.7, respectively). Between them the best monofuctional inducers was the [1,2]dithiole 12 with, like hybrid derivative 7, similar rates of QR specific activity in both cellular systems. In order to complete the study, for some of the most relevant monofunctional inducer derivatives, in other words, hybrids 5 and 7, and the unexpected

Table 2. Induction of quinone reductase activity on Hepa 1c1c7 and BPrc1 of the studied compounds, evaluated at 10 μM dose.

Compound

QR activity†

Compound

 

Hepa 1c1c7

BP c1

rH/B

1

1.20 ±0.01

1.70 ±0.02

2

1.61 ±0.01

1.21 ±0.02

3

1.17 ±0.01

4 5

QR activity†

 

Hepa 1c1c7

BPrc1

0.7

9

1.00 ±0.01

0.93 ±0.01 1.1

1.3

12

2.01 ±0.02

1.75 ±0.04 1.1

1.79 ±0.01

0.6

13

2.72 ±0.08

1.10 ±0.08 2.4

3.43 ±0.07

1.67 ±0.01

2.0

14

2.10 ±0.09

1.50 ±0.09 1.4

1.41 ±0.02

1.60 ±0.01

0.9

15

3.51 ±0.05

1.50 ±0.09 2.3

6

2.22 ±0.02

1.34 ±0.01

1.7

16

1.20 ±0.04

1.61 ±0.09 0.7

7

1.87 ±0.01

1.74 ±0.02

1.1

17

2.40 ±0.04

1.42 ±0.08 1.7

8

1.27 ±0.02

1.01 ±0.01

1.3

18

2.30 ±0.04

1.30 ±0.07 1.8

4.45 ±0.35

1.49 ±0.56

3.0

 

 

 

4BFV



r

§

rH/B §

 

Ratio of QR specific activity = QR specific activity of treated cells/QR specific activity of control cells. QR specific activity of control cells: for Hepa 1c1c7 line = 0.1400 ± 0.0013 U/mg protein, for BPrc1 line = 0.2200 ± 0.0063 U/mg protein. ‡ 4BFV: 4′-Bromoflavone. § rH/B: Ratio QR specific activity on Hepa1c1c7/on BPrc1. †

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Future Med. Chem. (2015) 7(7)

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Searching Phase II enzymes inducers, from Michael acceptor-[1,2]dithiolethione hybrids, as CCA 

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Table 3. Effects of selected compounds, 5, 7, 12, 16, and 18, on Hepa 1c1c7 and BPrc1 cytotoxic effects (IC50 ), induction of quinone reductase activity and chemopreventive indexes. Compound

Hepa 1c1c7†

BPrc1†

IC50 (μM)

CD (μM)

CI

5

>100.0 #

21.0

7

>100.0 #

12

26.0

16

>200.0

18

>200.0 #

4BFV t-BHQ



IC50 (μM)

IC50 (μM)

CD (μM)

CI §

>4.8

>100.0 #

19.1

>5.2

11.3

>8.8

>100.0 #

12.3

>8.1

10.0

2.6

51.0

26.0 7.5

85.0 147.0

#



§



26.0

2.0

>7.7

>200.0

#

25.0

>8.0

>26.7

>200.0 #

30.0

>6.7

0.79

107

150.0

80.0

1.9

1.0

147

60.0

6.0

10.0

Results are means of three independent experiments with standard deviation less than 10% in all cases. ‡ CD: concentration required to double the quinone-reductase § CI: ratio between IC50 and CD. ¶ Solubility problems in the biological milieu did not allow to probe higher doses. # t-BHQ: t-butylhydroquinone. CD: Quinone reductase activity; CI: Chemopreventive index.

containing compounds were bifunctional inducers, in other wrods, dithiolethione 14, dithioles 13 and 17, and salt 18; compounds with the Michael acceptor together with [1,2]dithiole system and without the structural features of (i) and (ii) were monofunctional inducers, i.e. hybrid derivatives 5 and 7, and the secondary product 12. Due to the presence of the polar phenolic OH and voluminous phenylcarbonylmethyl moieties seem to play a role in the biological properties, we analyzed theoretically some stereo-properties of the studied compounds in order to search for a relationship with the bioactivity. In this sense, as independent variables

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5 Ratio of specific GST activities

A ut h

or

Pr

is the fact that, unlike other Michael acceptors exhibiting cytotoxic effects, these new compounds showed lower toxicity than the reference compounds, 4BFV and t-BHQ, against studied cell lines (Table 3) . In order to complete the information about the capability of the studied compounds to induce phase II enzymes, we also studied the modification on GST in both cellular models (Figure 5) . Again, according to rH/B (defined for GST specific activity induction), the positive control, 4BFV, and the intermediate [1,2] dithiolethione 4 were classified as bifunctional inducers displaying higher GST specific activity on the wildtype cells, in other words rH/B of 2.7 and 2.2, respectively. Again, the hybrid derivative 6 was classified as bifunctional inducer (rH/B of 1.6). Similarly occurred with QR the hybrid compounds 5 and 7 were monofunctional inducers (rH/B of 0.8 and 1.0, respectively) being the best the cinnamate one, hybrid derivative 7. The induction capabilities to duplicate GST specific activity, CDs, for hybrid 7 was both in Hepa 1c1c7 and BPrc1 just under 10 μM consequently the CIs, expressed in terms of GST, were superior of 10.0 for both cellular models. This is in accordance with that obtained with QR demonstrating that the chemopreventive activity would be given by the induction of phase II enzymes. In order to explain the influence of the chemical structure on the desired biological activity we performed an initial structure-activity relationship (SAR) study (Figure 6) . Therefore, observing the group of studied compounds it could be drawn the following structural exigencies: –OH phenolic-moiety containing compounds were bifunctional inducers, in other words, parent chalcones 2 and hybrid 6, parent dithiolethione 4, and dithiole 13; phenylcarbonylmethyl-moiety

oo

f



Hepa1c1c7 BprC1

4 3 2 1 0 4BFV 1

2

3

4

5

6

7

9

12

Compound Figure 5. Induction of glutation S-transferase activity by the compounds, at 10 μM dose, on studied cellular models expressed as the ratio of glutation S-transferase specific activity. Ratio of GST specific activity = GST specific activity of treated cells/GST specific activity of control cells. GST specific activity of control cells: for Hepa 1c1c7 line = 0.1300 ±0.0081 U/mg protein, for BPrc1 line = 0.0650 ±0.0010 U/mg protein. 4BFV: 4′-Bromoflavone; GST: Glutation S-transferase.

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Research Article  Couto, de Ovalle, Cabrera, Cerecetto & González

O

O

HO

O

HO

HO

HO S

(1) Monofunctional

O

O

(3)

(2) Weak bifunctional

S

S

S S

S S

(4)

Monofunctional

Bifunctional

S

S

S

O

S

O

O

HO

O

O

O

O

(6)

Monofunctional

Bifunctional

O

Pr

O

S

(14)

or

O

O

O

S

S

A ut h S S

O

(13)

Strong monofunctional

S S

S S

(15), -R = -CH3 (17), -R = -Ph

O

Bifunctional

S

O

S

I

S

(16)

+

-

R

O

HO

S

S S

Bifunctional

Inactive

O

(12)

S

S S

(9)

Inactive

O

O

S

S S

(8)

Strong monofunctional

S S S

O

O O

(7)

oo

(5)

O

f

S

R O

Bifunctionals

S S

O

O

Monofunctional

I S

Ph

+

(18)

O

Bifunctional

Figure 6. Summary of biological behavior of the studied compounds in their ability to act as monofunctional or bifunctional inducers (using QR induction capability).

were employed theoretical volume, lipophilicities and molecular polar surface area (volume, miLogP and TPSA, respectively, Table 4) [33,34] , while rH/B was used as dependent variable. From these studies, we could observe that the bioactivity was related, in a quadratic manner, with lipophilicity or with volume of the active compounds (Figure 7) . Although the relationships were not statistically significant, considering in each study

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Future Med. Chem. (2015) 7(7)

some compounds are outliers, in other words derivatives 2 and 12 in the case of miLogP (Figure 7A) or derivatives 15 and 18 in the case of vol (Figure 7B), the correlation between rH/B and volume was the best (Figure 7B) . This result showed, as expected, that an optimum volume of the molecule could determine an effective interaction with biomolecules involved in the pathway of induction.

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Table 4. Some theoretical properties determined for the studied compounds. rH/B 

miLog P

TPSA

Volume (Å 3 )

1

0.7

3.722

54.37

288.46

2

1.3

3.662

74.598

298.68

3

0.6

3.574

9.234

238.55

4

2.0

3.038

20.228

217.81

5

0.9

7.259

43.376

478.92

6

1.7

7.199

63.604

489.13

7

1.1

5.211

26.305

367.35

8

1.3

4.663

26.305

352.71

9

1.1

3.119

26.305

284.55

12

1.1

3.157

60.447

415.67

13

2.4

6.28

54.37

14

1.4

4.722

26.305

15

2.3

3.612

16

0.7

-

17

1.7

6.816

18

1.8

-

f

Compound

483.72

oo

351.75

369.69

-

306.74

43.376

503.51

-

373.80

Pr

43.376

rH/B: Ratio QR specific activity on Hepa1c1c7/on BPrc1; TPSA: Theoretical molecular polar surface area. 

during 48 h on aqueous solutions of pH 2.0, 7.4 and 8.3 at 37°C. On the other hand, being one of the proposed mechanism of activation of Nrf2 by [1,2]dithiolethione the release of H2S [19] we also studied the stability of the selected derivatives in the culture milieu of the biological assay, specially looking for some selected metabolites related to this release process. In this sense, intermediate 3 and salt 16 were incubated during 48 h in the presence of Hepa 1c1c7 cells and apparition of [1,2]dithiole3-one 19 [37] was checked (Figure 9) . Neither apparition of 19 from thione 3 and salt 16 nor the transformation of 16 to 3 were evidenced (see the corresponding HPLC-chromatograms in Supplementary Material section). Proving that the proposal shown in Figure 9 was not operative for this pair of compounds.

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or

Due to the existence of outliers and different group of biological behaviors, mono- and bifunctional inducers, we could speculate that other properties could be involved in the bioresponses. Specifically in the case of bifunctional agents, the presence of outliers could be the result of different induction pathways. For this reason, we also analyzed the electronic properties of the most relevant compounds, the best monofunctional inducer derivatives, in other words 7 and 12, and the bifunctional one, in other words 13. After the analyses of the different maps of charge distribution, we observed that monofunctional inducer derivatives share the LUMO maps (Figure 8) . We could observe that these compounds possess a region that could clearly act as Michael acceptor systems; however, derivative 13, one of the best bifunctional inducer derivative, lacks of this feature. Probably this property could also play a role in the bioactivity. Considering the great potential therapeutic use of this class of compounds, it is indispensable for pharmaceutical development to assess their stability [35] . The hybrids could hydrolyze via the ester moiety. In order to confirm this proposal, we analyzed the stability of hybrid cinnamate 7 on aqueous solution at different pHs and in the presence of rat plasma. The compound was stable on plasma for 30 min at 37°C, after this time cinnamic acid and phenol 4 were evidenced. The half-life of compound 7 in plasma was 60 min at 37°C. There are current drugs having similar half-life values in plasma [36] as cinnamate 7. Additionally, compound 7 was stable

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Experimental Chemistry

Reagents were purchased from Aldrich and used without further purification. Melting points were performed using an Electrothermal Engineering Ltd melting point apparatus, and the results were uncorrected. 1H and 13 C NMR spectra were recorded in the indicated solvent on Bruker DPX 400 MHz spectrometer. Chemical shifts were quoted in parts per million downfield from TMS and the coupling constants were expressed in Hertz. Mass spectra were recorded on a Hewlett Packard MSD 5973 (electronic impact [EI]) or on a Hewlett Packard LC/MS Series 1100 (electrospray ionization, ESI) instruments. All solvents were dried and

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Research Article  Couto, de Ovalle, Cabrera, Cerecetto & González gen atmosphere. 5-(4-Hydroxyphenyl)-3H-[1,2]dithiol3-thione (4), dicyclohexyl­ carbodiimide (1.3 equiv.), and a catalytic amount of 4-dimethylamino­ pyridine (0.1 equiv.) was added to the mixture. After stirring until the absence of 4 (checked by TLC) the mixture was filtered off to remove dicyclohexylurea and the remaining solution was washed with HCl (0.1 M, 2 × 20 ml) and NaOH (0.1 M, 2 × 20 ml). The organic layer was dried over Na2SO4, evaporated in vacuo and purified by column chromatography (SiO2, CHCl2:n-hexane 1:1).

rH/B

A 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4

Bifunctional derivatives Outliers

Monofunctional derivatives 3

4

5 6 miLog P

8

oo Pr

Bifunctional derivatives Outliers

Monofunctional derivatives

200

250

or

rH/B

2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4

300

350

400

450

500

550

Vol (A3)

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Figure 7. Structure–activity studies. (A) rH/B versus calculated log P. (B) rH/B vs calculated volume. The curves show only tendencies. In red appear outlier compounds for each group of study. rH/B: Ratio QR specific activity on Hepa1c1c7/on BPrc1.

distilled prior to use. All the reactions were carried out in a nitrogen atmosphere. Reactions were monitored by TLC using commercially available precoated plates (Merck Kieselgel 60 F254 silica) and developed plates were examined under UV light (254 nm) or as iodine vapor stains. Column chromatography was performed using 200 mesh silica gel. The purity of the compounds were determined by microanalyses on a Fisons EA 1108 CHNS–O instrument from vacuum-dried samples and were within ±0.4 of the values obtained by calculated compositions. Intermediates 1–4 and 9, secondary and unexpected products 12–18 were prepared following synthetic procedures previously reported [26,27,38,39] . General procedure for the preparation of hybrid derivatives 5–8

In a 50 ml round-bottom flask, a mixture of the corresponding acid (1 equiv.) and dichloromethane (15.0 ml per 0.3 mmol of 4) was cooled in an ice-bath under nitro-

866

Reaction time: 6 h. Yield: 24%; Orange solid; mp 198–201°C. 1H NMR (CDCl 3), δ: 8.27 (d, 2H, J=8.3), 8.08 (d, 2H, J=7.1), 7.88 (d, 1H, J=15.8), 7.82 (d, 2H, J=8.3), 7.76 (dd, 2H, J=12.8), 7.69 (d, 1H, J=15.7), 7.64 (dt, 1H, J1=2.6, J2 =1.7), 7.56 (t, 2H, J=7.5), 7.46 (s, 1H), 7.42 (d, 2H, J=8.8). 13CNMR (CDCl3), δ: 123.1, 124.6, 125.1, 128.3, 128.7, 128.9, 129.1, 129.4, 130.7, 134.2, 135.4, 137.2, 142.1, 142.8, 152.8, 163.8, 171.3, 189.9, 215.7. MS (EI), m/z (%): 460 (M +., 6), 235 (100), 226 (7), 207 (12), 178 (15), 105 (7), 77 (9). Calculated for C25H16O3S3 : C, 65.2; H, 3.5; S, 20.9. Found: C, 64.9; H, 3.3; S, 21.1.

f

B

7

5-{4-[4-(3-Oxo-3-phenyl-1E-propenyl)] benzoyloxy]phenyl}-3H-[1,2]dithiole-3-thione (5)

Future Med. Chem. (2015) 7(7)

5-{4-[4-(3-(2-Hydroxyphenyl)-3-oxo-1Epropenyl)]benzoyloxy]phenyl}-3H-[1,2]dithiole-3thione (6)

Reaction time: 48 h. Yield: 6%; Orange solid; mp 184–187°C. 1H NMR (CDCl3), δ: 12.7 (s, 1H), 8.29 (d, 2H, J=8.3), 8.10 (d, 1H, J=8.7), 8.02–7.94 (m, 2H), 7.88–7.73 (m, 4H), 7.57 (t, 1H, J=7.7), 7.47 (s, 1H), 7.42 (dd, 1H, J1=8.5, J2 =2.8), 7.38 (d, 1H, J=8.6), 7.09 (d, 1H, J=8.2), 7.01 (t, 1H, J=7.7). 13CNMR (CDCl3), δ: 118.8, 119.3, 123.2, 127.7, 128.6, 129.4, 129.5, 129.6, 130.9, 134.5, 135.9, 139.2, 140.9, 143.4, 153.9, 162.3, 163.8, 171.9, 193.1, 215.2. MS (EI), m/z (%): 476 (M+., 20), 460 (19), 226 (94), 161 (100). Calculated for C25H16O4S3 : C, 63.0; H, 3.4; S, 20.2. Found: C, 62.8; H, 3.1; S, 20.2. 5-[4-(3-Phenyl-2E-propenoyloxy)phenyl]-3H-[1,2] dithiole-3-thione (7)

Reaction time: 8 h. Yield: 80%; Orange solid; mp 151–153°C. 1H NMR (CDCl3), δ: 7.93 (d, 1H, J=16.0), 7.75 (d, 2H, J=8.7), 7.63 (dd, 2H, J1=6.6, J2 =3.2), 7.50–7.43 (m, 4H), 7.36 (d, 2H, J=8.5), 6.66 (d, 1H, J=16.0). 13C-NMR (CDCl3), δ: 116.3, 123.0, 127.9, 128.0, 128.3, 128.5, 129.1, 133.9, 136.0, 147.7, 151.7, 164.8, 171.8, 215.5. MS (EI), m/z (%): 356 (M+., 15), 226 (2), 161 (3), 132 (10), 131 (100), 103 (31), 77 (12). Calculated for C18H12O2S3 : C, 60.6; H, 3.4; S, 27.0. Found: C, 60.5; H, 3.3; S, 26.8.

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Searching Phase II enzymes inducers, from Michael acceptor-[1,2]dithiolethione hybrids, as CCA 

Research Article

A

7

13

12

B

5-[4-(2-Phenylethanoyloxy)phenyl]-3H-[1,2] dithiole-3-thione (8)

(FBS), respectively, and grown at 37°C under a 5% CO2–95% air atmosphere. Preparation of cytosolic fraction and assay procedure

Pr

Reaction time: 7 h. Yield: 38%; Orange solid; mp 136–139°C. 1H NMR (CDCl3), δ: 7.68 (d, 2H, J=8.7), 7.43–7.40 (m, 6H), 7.24 (d, 2H, J=8.8), 3.92 (s, 2H). 13C-NMR (CDCl3), δ: 40.0, 118.1, 122.6, 122.8, 127.3 127.6, 127.8, 128.2, 128.9, 129.3, 130.4 132.9, 136.1, 153.6, 169.5, 171.9, 215.3. MS (EI), m/z (%): 344 (M+., 23), 226 (11), 120 (9), 132 (8), 77 (6). Calculated for C17H12O2S3 : C, 59.3; H, 3.5; S, 27.9. Found: C, 59.7; H, 3.1; S, 28.2.

oo

f

Figure 8. Theoretical studies. (A) Representation of the studied molecules in ball and spoke. Colors for the atoms are the commonly used. (B) Lowest unoccupied molecular orbital surfaces (red, isovalue: 0.0082) and electronic density surfaces (gray, isovalue: 0.002). In blue are highlighted the presence of Michael acceptors in monofunctional derivatives and in black the lack of this system for the bifunctional derivative. For color images please see online at: www.future-science.com/doi/full/10.4155/FMC.15.32.

or

A suspension containing 8.0 × 104 cells in 1.0 ml of milieu was sown in 24-well plate and incubated 24 h to allow for cell attachment. The milieu was aspirated and the cells washed twice with 1.0 ml phosphate buffered saline (PBS). Compounds were tested by triplicate at the desired concentration, for the initial study at 10 μM, for the CD (concentration required to double QR activity) determination at 2.5, 5.0, 8.0, 10.0 and 20.0 μM, in 1.0 ml of fresh milieu and not exceeding 0.5% v/v DMSO. The cells were treated for 48 h, milieu was aspirated, cells washed twice with PBS and then detached using 0.1 ml trypsin/EDTA. Fresh milieu was added and 1.0 ml of cellular suspension was transferred into 1.5 ml tubes and centrifuged at 10,000 g for 5 min. The milieu

Biology

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Cell culture and conditions

Mouse hepatoma Hepa1c1c7 cell (ATCC, CRL-2066) and its mutant BPrc1 (ATCC, CRL-2217) were purchased from American Type Culture Collection (ATCC, VA, USA). They were cultured in α-MEM and DMEM containing 10% fetal bovine serum

S

O

S

S

O

S

S

S S

O

O

S

(3)

(19) Metabolism?

S O

S

S

O S

(16)

I

S S

+

-

Outside the cell

O

I

+

-

O

Inside the cell

Figure 9. Speculative metabolism of [1,2]dithioles 3 and 16.

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Research Article  Couto, de Ovalle, Cabrera, Cerecetto & González

Assay of QR activity

Stability studies [35]

For the determination of plasma stability, we used a pool of healthy rat plasma diluted 1:1 in sterile Tris. HCl buffer, pH 7.4. The stock solution of compound 7 was prepared at 10 mM in DMSO, and the final concentration in the biological milieu was 40 μM (DMSO